A DNA molecule containing a gene which reverses a mutant phenotype of a strain of Saccharomyces cerevisiae is isolated and purified. The gene is GLS1 (glucan synthesis gene 1). GLS1 encodes a subunit of 1,3-.beta.-D glucan synthase. The protein encoded by GLS1 represents a target for drug therapy for fungal disease. The invention includes homologues of GLS1 is isolated from other fungi, such as Aspergillus fumigatus, Candida albicans, Schizosaccharmomyces pombe and Phytophthora infestans.
Understanding the mode of action of therapeutic compounds requires a variety of experimental approaches. One approach involves the isolation of organisms resistant or sensitive to test compounds. Such organisms may be used to isolate genes encoding the drug targets.
The fungal cell wall is a complex structure composed of a number of polymers: chitin, .alpha.- and .beta.-glucans, and mannoproteins. The fungal cell wall is involved in a variety of vital cellular processes: vegetative growth, morphogenesis, uptake and secretion of macromolecules and protection against osmotic changes are affected by changes in the composition and integrity of the cell wall. Antifungal compounds which act via the inhibition of cell wall synthesis (a process essential to fungi and absent from mammalian cells) may have high fungicidal activity and low toxicity to mammalian cells.
One class of .beta.-glucan inhibitors is comprised of lipopeptide antibiotics such as aculeacin A, echinocandin B and the pneumocandins. These compounds are cyclic hexapeptides that contain a non-polar fatty acid side chain. Echinocandins are fungicidal because they inhibit synthesis of 1,3-.beta.-D glucan, which disrupts the integrity of the cell wall and causes lysis of yeast cells. In vitro echinocandins inhibit polymerization of glucose into 1,3-.beta.-D glucan.
Another class of .beta.-glucan synthesis inhibitors comprises the papulacandins and chaetiacandin. These compounds contain a glycoside component connected to an aromatic ring system and two long chain fatty acids. These compounds have the same mode of action as the echinocandins.
It has been shown that Pneumocystis carinii has .beta.-glucan in the wall of its cyst form (Matsumoto, Y., et al., 1989, J. Protozool. 36: 21S-22S). Inhibitors of .beta.-glucan synthesis, such as papulacandins and echinocandins, may be useful in the treatment of P. carinii infections. In a rat model of P. carinii pneumonia, L-671,329 (an echinocandin) and L-687,781 (a papulacandin) were both effective in reducing the number of cysts in the lungs of infected rats (D. M. Schmatz et al., 1990, PNAS 87: 5950-5954). These results suggest that .beta.-glucan synthesis is a target for the identification of therapeutics useful in the treatment of P. carinii infections.
There have been a number of efforts to isolate drug-resistant yeast strains affected in .beta.-glucan synthesis. The mutants that have been isolated include acul (Mason, M. M., et al., 1989, Cold Spring Harbor Laboratory, Abstract #154), and pap1 (Duran, A., et al., 1992, Profiles in Biotechnology (T. G. Villa and J. Abalde, Eds.) Serivicio de Publicaciones, Universidad de Santiago, Spain. pp. 221-232).
In the present work a more potent echinocandin (L-733,560) was used as a selective agent to isolate mutant strains specifically affected in glucan synthesis. One mutant (strain MS14) is echinocandin-resistant and is also supersensitive to the chitin synthase inhibitor nikkomycin Z. The mutation in MS14 maps to the FKS1 gene and is designated fks1-4. Another mutant (strain MS1) is resistant to echinocandins and supersensitive to both papulacandin and rapamycin. Strain MS1 was used to clone the GLS1 gene.